The principle of obtaining embryos by culturing somatic cells in vitro is well known, on paper. In particular, see D. J. Gray and J. A. Mortensen, Plant Cell, Tissue and Organ Culture (1987) vol. 9 pp. 73-80. Regarding vine somatic embryos in particular, see U.S. Pat. No. 4,532,733. While somatic embryogenesis may be feasible for certain species of plants, this method is, by contrast, very difficult, if not impossible, to implement in the case of other species.
In order to understand the problems which are posed, it is necessary to recall briefly the principles of culturing somatic cells which are intended to form embryos.
Somatic embryogenesis essentially comprises two steps:
1--the induction of embryogenic potential using exogenous auxins, in general at high concentrations, which give rise to the appearance of proembryogenic aggregates (or masses) (PEM), which correspond to groups of from 10 to 50 cells which are, in particular, of a meristematic nature. PA1 2--the transfer of these cells onto media which lack auxins and which lead to the formation of somatic embryos from these PEM, following the different developmental stages of plant embryogenesis, namely: globules, heart and torpedo.
In the case of certain plant species, it proves to be very difficult, if not impossible, to obtain somatic embryos. For example, in the case of certain vine cultivars, the embryogenic capacities of the somatic cells can either be halted, usually at the stage of proembryogenic aggregate formation, or else maturation of the embryos is blocked, in particular at the globular stage.
This is all the more unfortunate since there exists, in particular in the case of the vine, a very great need for obtaining somatic embryos, in vitro, on a large scale. Thus, the in vivo methods for producing embryos are often inadequate and undesirable since they lead, as a general rule, to genetic recombinations which involve the loss of the novel genetic features of the vine variety or the stock-vine whose multiplication is desired.
European patent application EP 281375 proposed adding calmodulin and calcium to a cell culture in order to promote differentiation of roots and embryos.
European patent application EP 455597 describes a method for stimulating the growth and embryogenesis of plants cultivated in vitro, by addition of arabino-galactan proteins. However, it has been demonstrated (de Vries et al., 1989, Proceedings of the International Symposium of Biotechnology for Major Crops (A.D.E.B.I.O. ed.), pages 22-23) that an extracellular glycoprotein of 52/54 kDa induces arrest in embryonic development at the heart stage.